This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this proposal is to define the role of pro-metastatic protein HEF1 in breast cancer metastasis to mouse orthotopic models. Development of xenograft models is justified based on our need to identify the steps of metastasis affected by HEF1 to develop therapeutic strategies for advanced stage breast cancer patients where HEF1 is commonly overexpressed. We have established several highly metastatic breast cancer cell lines with regulated expression of HEF1 and shRNA against HEF1 to directly test its action in promoting cell migration, invasion and proliferation in mouse tumor models. Subsequently, we will define how HEF1 downstream targets, Aurora-A and HDAC6, affect the regulation of mitotic progression and invasion of metastatic breast cancer cells. In Aim 1, we will assess the consequences of HEF1 depletion/or overexpression in metastatic breast cancer cells in vivo, by injecting HEF1 -manipulated tumor cells into the mammary fat pads of SCID mice. We will follow tumor growth, dissemination, and distant sites colonization steps using non-invasive multimodal imaging techniques. In Aim 2 we will assess the role of HEF1 activated proteins AurA and HDAC6 in metastasis and evaluate efficacy of AurA and HDAC6 inhibitors currently in clinical trials for metastatic breast cancer treatment based on the expression of HEF1 in the tumor cells. This analysis will enable us to determine the impact of HEF1 expression at each stage of metastasis, define the role of AurA and HDAC6 in HEF1-driven processes, and develop the mechanistic rationale for future drug development.